Ferritin Cage Enzyme Encapsulation as a New Platform for Nanotechnology

Reporter: Irina Robu, PhD

In bionanotechnology, biological systems such as viruses, protein complexes, lipid vesicles and artificial cells, are being developed for applications in medicine and materials science. However, the paper published by Stephan Tetter and Donald Hilvert in Angewandte Chemie International Edition show that it is possible to encapsulate proteins such as ferritin by manipulating electrostatic interactions with the negatively charged interior of the cage.The primary role of ferritin is to protect cells from the damage caused by the Fenton reaction; where, in oxidizing conditions, free Fe(II) produces harmful reactive oxygen species that can damage the cellular machinery.

The ferritin family proteins are protein nanocages that evolved to safely store iron in an oxidizing world. Since ferritin family proteins are able to mineralize and store metal ions, they have been the focus of much research for the production of metal nanoparticles and as prototypes for semiconductor production. The ferritin cage itself is highly symmetrical, and is made up of 24 subunits arranged in an octahedral symmetry. Ferritins are smaller than other protein used for protein encapsulation. Their outer diameter is only 12 nm, whereas engineered lumazine synthase variants form cages with diameters ranging from about 20 to 60 nm.The ferritin cage displays remarkable thermal and chemical stability it is likely to modify the surface of the ferritin cage through the addition of peptide and protein tags. These characteristics have made ferritins attractive vectors for the delivery of drug molecules and as scaffolds for vaccine design.

In summary, the paper published in Angewandte Chemie International Edition is the first example of protein incorporation by a ferritin. Dr. Donald Hilvert and colleagues have shown that AfFtn not only complexes positively charged guest proteins within its naturally negatively charged luminal cavity, but that the in vitro mixing technique can be extended to the encapsulation and protection of other functional fusion proteins.

Hence, the recent discovery of encapsulated ferritins has identified an exciting new platform for use in bio nanotechnology. The use of synthetic biology tools will allow their rapid implementation in materials science, bio-nanotechnology and medical applications.

Scientists at Indiana University (IU) have identified a genetic mechanism that is likely to drive mutations that can lead to cancer. Their E. coli study, published in the Proceedings of the National Academy of Sciences, finds the enzyme APOBEC3G, a known trigger for mutations that occur as benign tumor cells to transform into cancerous malignancies that spread throughout the body, appears to cause these harmful changes by mutating genes during DNA replication.

The study also received support from the Wayne State University School of Medicine, whose researchers provided expertise on APOBEC3G and helped analyze the data. All experiments were carried out at IU.

“Many tumors accumulate mutations during their growth, which lead to the subsequent characteristics that permit metastasis,” said Patricia Foster, Ph.D., the principal investigator on the grant and a professor in the IU Bloomington College of Arts and Sciences’ biology department, senior author on the study. “Based upon the results revealed in bacteria in our study, we believe that the APOBEC family of enzymes creates some of these mutations specifically during the rapid growth of these tumors.”

The results could have implications for personalized medicine. For example, because it is possible to identify tumors potentially vulnerable to the enzyme by using current DNA sequencing technology, a physician treating these tumors might want to explore temporarily suppressing expression of this enzyme, she said.

Normally, the APOBEC family of enzymes plays an important role in the human immune system by driving changes in immune cells that aid in defense against viruses, possibly including the HIV/AIDS virus. The IU scientists found the harmful influence of the enzyme family arises from the complex way that two halves of every double-stranded DNA molecule must unravel to replicate during cellular division—splitting into two temporarily single-stranded DNA chains thousands of links (the four nucleotides) in length to serve as templates for the new copy. As the nucleotides are split in half to be copied, one of the two single-stranded bits of DNA, known as the lagging strand template, is highly vulnerable to genetic mutation, according to Dr. Foster.

This “gap in the armor” occurs because DNA polymerase must repeatedly traverse the nucleobases in the lagging strand template thousands of times during the course of replication, stopping further down the chain from the base pair previously inserted on the loop along the chemical chain. Each of these polymerase “hops” creates a long stretch of DNA that temporarily remains as a single strand.
The complex process introduces more opportunities for errors in the lagging strand template compared to the continuous step-by-step process that replicates the other half of the split strand of DNA, called the leading strand template.

“We’re talking about thousands of bases exposed without a complimentary strand throughout the whole replication cycle,” noted Dr. Foster. “If I were going to design an organism, I would make two types of copying enzymes. An important organism for studying genes, E. coli allows scientists to observe genetic changes over thousands of generations in a relatively short time span. The results apply to humans as well as bacteria since the basic mechanisms of DNA replication are the same across all species.”

The mechanism by which the APOBEC family of enzymes drives mutation is cytosine deamination, in which a cytosine, the C nucleotide, transforms into uracil, one of the four bases in RNA that doesn’t play a role in DNA replication. But the presence of uracil during DNA replication can cause an error when a thymine, the T nucleotide, replaces a cytosine. APOBEC enzymes specifically target the C’s in single-stranded DNA for deamination.

The disruptive effect of the enzyme on genetic replication in the study was observed in a strain of E. coli, whose ability to remove the dangerous uracils had been switched off. To conduct the experiment, Dr. Foster’s lab observed the effect of APOBEC3G on approximately 50 identical lineages of E. coliover the course of nearly 100 days, with each day encompassing 20 to 30 bacterial generations.

Over time, a unique pattern of nucleotides was detected in the mutated DNA, a chain of three cytosine molecules, the same genetic signature found in other studies of the enzyme family. And these mutations were four times more likely to be found on the lagging-strand template than on the leading-strand template.

“These results strongly suggest that these mutations occur as APOBEC3G attacks cytosines during DNA replication, while they’re most exposed on the lagging strand template,” Dr. Foster said. “This basic mechanism appears to be the same in bacteria and in human tumors cells.”

Research on the effect of the enzyme APOBEC3G on DNA replication was conducted in the bacteria Escherichia coli. (Photo: Department of Defense)

A key group of enzymes could be the “gap in the armor” of all DNA, allowing cancer-causing mutations, according to a new study.

APOBEC3G, which is known to trigger benign mutations, also causes malignant mutations during the DNA replication process, according to the new findings, in the Proceedings of the National Academy of Sciences.

“Many tumors accumulate mutations during their growth, which leads to the subsequent characteristics that permit metastasis,” said Patricia Foster, professor at Indiana University, and senior author. “Based upon the results revealed in bacteria in our study, we believe that the APOBEC family of enzymes create some of these mutations specifically during the rapid growth of these tumors.”

The investigators created and observed the mutations in the bacteria Escherichia coli, which presented the advantage of watching thousands of generations in a relatively short time.

The key process is the movement of DNA polymerase along one of the two DNA single strands, known as the lagging strand template, during the replication process. The lagging strand becomes susceptible to errors. APOBEC can enter into this process, causing cytosine deamination, essentially replacing the intended cytosine on the strand with the thymine nucleobase, causing the mutations.

The scientists turned off the ability to regulate the cytosine deamination in the E. coli replication – and then observed an uptick in the harmful mutations confirming the culprit, they said.

“These results strongly suggest that these mutations occur as APOBEC3G attacks cytosines during DNA replication, while they’re most exposed on the lagging strand template,” said Foster. “This basic mechanism appears to be the same in bacteria and in human tumor cells.”

The study was supported in part of a $6.2 million grant from the U.S. Army Research Office to investigate bacterial evolution, according to the school.

The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation ofDnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.

Cytidine (C) to Uridine (U) RNA editing is a post-trancriptional modification that until recently was known to only affect Apolipoprotein b (Apob) RNA and minimally require 2 components of the C to U editosome, the deaminase APOBEC1 and the RNA-binding protein A1CF. Our latest work has identified a novel RNA-binding protein, RBM47, as a core component of the editosome, which can substitute A1CF for the editing of ApoB mRNA. In addition, new RNA species that are subjected to C to U editing have been identified. Here, we highlight these recent discoveries and discuss how they change our view of the composition of the C to U editing machinery and expand our knowledge of the functional attributes of C to U RNA editing.

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies.

MicroRNAs (miRNAs)2 are 20-22-nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages, including plants, insects, vertebrate, and mammals (1–3). More than 474 miRNAs have been identified in humans so far, and ∼30% of the genes in the human genome are predicted to be subject to miRNA regulation (4). The expression of many miRNAs is usually specific to a tissue or developmental stage, and the miRNA expression pattern is altered during the development of many diseases (3). Mature miRNAs are generated from RNA polymerase II-transcribed primary miRNAs that are processed sequentially by the nucleases Drosha and Dicer. Although miRNA can guide mRNA cleavage, the basic function of miRNA is to mediate inhibition of protein translation (1, 5–8) through miRNA-induced silencing complexes (miRISCs). The guiding strand of miRNA in a miRISC interacts with a complementary sequence in the 3′-untranslated region (3′-UTR) of its target mRNA by partial sequence complementarities, resulting in translational inhibition (1). A 7-nucleotide “seed” sequence (at positions 2-8 from the 5′-end) in miRNAs seems to be essential for this action (4). The composition of the miRISC is similar to that of the RNA-induced silencing complex (RISC), which is responsible for mRNA cleavage guided by small interfering RNAs (siRNAs) (1, 3, 7). Nevertheless, some differences exist between miRISCs and siRNA RISCs. For example, the major Argonaute protein in siRNA RISC is Ago-2, whereas all four of the Ago proteins (Ago1-4) are found in miRISC (3, 8). Further, the siRNA RISC may be associated with various RNA-binding proteins such as fragile-X mental retardation protein (FMRP), TAR RNA-binding protein (TRBP), and the human homolog of the Drosophilahelicase Armitage, Mov10, possibly in a cell type-specific manner (9–13).

The miRNA-mediated translational repression consistently correlates with an accumulation of miRNA-bound mRNAs at cytoplasmic foci known as processing bodies (P-bodies) (8). Several lines of evidence have indicated that P-bodies are actively involved in miRNA-mediated mRNA repression (14). The P-body-associated protein GW182 associates directly with Ago-1 (15, 16). Depletion of P-body components such as GW182 and Rck/p54 prevents translational repression of target mRNAs (8, 14–19). Furthermore, several miRISC-related components, such as miRNAs, mRNAs repressed by miRNAs, Ago-1, Ago-2, and Mov10, are found in P-bodies (14). P-body formation is a dynamic process that requires continuous accumulation of repressed mRNAs (20). However, P-bodies serve not only as sites for RNA degradation, but also for storage of repressed mRNAs (15). These mRNAs may later return to polysomes to synthesize new proteins (14). In fact, some cellular proteins can facilitate the exit of miRNA-bound mRNAs from P-bodies. For example, a stress situation may induce the relocation of HuR, an AU-rich element-binding protein, from the nucleus to P-bodies in the cytoplasm where it binds to the 3′-UTR of its target mRNA encoding CAT-1 (21). This binding increases the stability of the miR-122-bound mRNA by assisting it to egress from the P-body and return to polysomes. However, the mechanism underlying this reverse transport of miRNA-bound mRNA out of P-bodies remains to be further clarified.

The cellular apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G protein (APOBEC3G or A3G) is a potent antiretroviral factor that belongs to the cytidine deaminase family (22, 23). A3G can be incorporated into HIV-1 particles and cause extensive C to U conversion in the viral minus-stranded DNA during reverse transcription (24–26), which can trigger its degradation by virion-associated uracil DNA glycosylase-2 (UNG2) and apurinic/apyrimidinic endonucleases (APE) or lethal hypermutation in the HIV-1 genome (26, 27). However, accumulating evidence indicates that A3G protein carrying mutations in the catalytic domain of the cytidine deaminase retains substantial anti-HIV-1 activity (24, 28–31). Interestingly, A3G is found in P-bodies and stress granules (32, 33). It is associated with a high molecular mass structure (>700 kDa) in replicating cells, and this interaction is RNase-sensitive (34, 35). Further studies indicate that A3G interacts with many RNA-binding proteins, among which are several miRNA-related proteins, such as Ago1, Ago2, Mov10, and poly(A)-binding protein 1 (PABP1). These interactions are either partially or completely resistant to RNase A digestion (32, 35, 36).3 Aside from its inhibitory function in relation to endogenous retroviruses and other retrotransposons (37–41), the major cellular function of A3G seems to be related to P-body-related RNA processing and metabolism. As recent development has indicated that the function of P-body is closely related to miRNA activity, we therefore investigated the possibility of a connection between A3G and miRNA function.

A3G Counteracts miRNA-mediated Repression of Protein Translation—We first examined the effect of A3G on the expression of miRNAs. Using a miRNA microarray method, we did not find that A3G significantly changed the miRNA expression in 293T cells (supplemental Figs. S1 and S2). A3G also did not significantly change the expression of miRNA processors such as Drosha and Dicer1 or RISC components such as Ago2 and Mov10 (supplemental Fig. S3). Further, A3G also did not change the level of expression of P-body components such as GW182, Xrn1 and Lsm1 (supplemental Fig. S3). Nevertheless, the microarray data did indicate that several miRNAs, such as mir-16, mir-10b, mir-25, and let-7a, are abundant in 293T cells.

To study whether A3G affects the efficiency of miRNA-mediated translational repression, various 293T cell-enriched miRNA-binding sites with perfect or partial complementarity to their corresponding miRNAs were inserted into the 3′-UTR of luciferase (luc) or gfp (Fig. 1a). These plasmids were transfected into 293T cells, which naturally do not express A3G (22, 27), with or without an A3G-HA-expressing plasmid. Fig. 1b shows that the presence of mir-16, mir-10b, or mir-25 miRNA-binding sites in the 3′-UTR of luc gene remarkably inhibited the expression of luciferase. Interestingly, A3G significantly counteracted this inhibition. Similar phenomenon can be observed in HeLa cells (Fig. 1c). To verify this derepression, a dose dependence experiment was performed and derepression was found to correlate with the A3G expression level (Fig. 1d). Real-time PCR data showed that the expression level of luciferase mRNA also substantially increased concomitantly with the expression level of A3G (Fig. 1e). This derepression of miRNA-mediated translational inhibition still occurred when the reporter gene was changed to gfp (Fig. 1f).

A3G counteracts miRNA-mediated repression of protein translation in 293T and HeLa cells.a, sequences of the miRNAs mir-16, mir-25, mir-10b, and let-7a and their target sites used for reporter gene constructs are shown. b and c, 293T cells (b) or HeLa cells (c) were co-transfected with a plasmid expressing A3G (pcDNA-A3G-HA) and a plasmid containing the luciferase reporter gene with binding sites for mir-16, mir-10b, or mir-25 in the 3′-UTR. pcDNA3 and pmir-REPORT were also transfected as controls. At 48-h post-transfection, luciferase activity was measured. d and e, 293T cells were co-transfected with different amounts of A3G-expressing plasmid (ranging from 0 to 0.4 μg) and pmir16-luc. At 48-h post-transfection, luciferase activity (d) was measured, and luciferase mRNA (e) was detected by real-time RT-PCR. The means ± S.D. are shown. f, 293T cells were co-transfected with pcDNA-A3G-HA and a plasmid containing a GFP reporter gene with a binding site for let-7a in the 3′-UTR, pEGFP-c1-let-7a, or pEGFP-c1 as a control. At 48-h post-transfection, GFP expression was analyzed by FACS and the mean fluorescence intensity (MFI) of GFP was determined. The data shown are representative of at least three replicates.

A3G/F-specific siRNA restores miRNA-mediated repression of protein translation in A3G/F-rich T-lymphocytes and macrophages. PHA-activated CD4+ T cells (a) and H9 cells (b) were first transfected with A3G- and A3F-specific siRNAs via Nucleofector (AMAXA). A siRNA for luc was used as a control for transfection. After 48 h, the cells were transfected with pEGFP-c1-let-7a or pEGFP-c1. At 48-h post-transfection, GFP expression was analyzed by FACS. The MFI of GFP from pEGFP-c1 was set as 100%. The means ± S.D. are shown. c, primary monocyte-derived macrophages were first transfected with A3G- and A3F-specific siRNAs. A siRNA for luc was used as a control for transfection. After 48 h, the cells were transfected with pEGFP-c1-let-7a or pEGFP-c1. pcDNA3-A3G-HA (2 μg) was also cotransfected for overexpression experiment. At 48-h post-transfection, GFP expression was analyzed by Western blotting analysis via anti-GFP antibody. The expression of A3G and A3F were also examined by Western blotting.

APOBEC3 family members inhibit miRNA-mediated repression of protein translation. 293T cells were co-transfected with plasmids expressing APOBEC3 family members and pmir16-luc (a) or with plasmids expressing various A3G mutants and pmir16-luc (b). At 48-h post-transfection, luciferase activity was measured. The 4C mutant represents an A3G mutant that has four point mutations: C97A/C100A/C288A/C291A. The means ± S.D. are shown.

Furthermore, to confirm this effect, H9 T-cells, PHA-activated primary CD4+ T-lymphocytes and macrophages, which naturally harbor significant amounts of A3G and another APO-BEC3 protein, A3F, were treated with A3G- and A3F-specific siRNAs. Western blotting showed that expression of A3G and A3F could be effectively decreased by these siRNAs (Fig. 2, a-c). The depletion of A3G and A3F enhanced the efficiency of let-7a miRNA-mediated translational repression in these A3G/F-enriched cells (Fig. 2, a-c). Conversely, overexpression of A3G/F in macrophages can substantially enhance the derepression of miRNA-mediated translational inhibition (Fig. 2c, lane 1).

Other APOBEC3 Family Members Also Inhibit miRNA-mediated Repression of Protein Translation—To test whether other APOBEC3 family members also regulate miRNA repression, vectors expressing the APOBEC3 family members A3B, A3C, and A3F were transfected into 293T cells. All the tested APOBEC3 family members were able to inhibit the miRNA-mediated translational repression (Fig. 3a). Interestingly, a synergistic effect was found between various APOBEC3 family members (Fig. 3a).

A3G enhances the association of mir-16-targeted mRNA with polysomes. 293T cells were co-transfected with pMIR-REPORT and pcDNA3 (a), pMIR-REPORT and pcDNA3-A3G (b), pmir16-luc and pcDNA3 (c), pmir16-luc and pcDNA3-A3G (d), pmir16-luc and anti-mir16 inhibitors (e), or pmir16-luc and anti-mir28 inhibitors (f). At 48-h post-transfection, polysome profile analysis was performed and the distribution of luciferase mRNA and β-tubulin mRNA in the fractions was analyzed by RT-PCR. 293T cells were co-transfected with pmir16-luc and pcDNA3-A3G (g), or pMIR-REPORT alone (h). Prior to collection, the cells were treated with puromycin (0.3 mg/ml) for 30 min. At 48-h post-transfection, polysome profile analysis was performed, and the distribution of luciferase and β-tubulin mRNA in the fractions was analyzed by RT-PCR.

Given that A3G has cytidine deaminase activity, we examined whether this activity is responsible for the A3G inhibitory effect on miRNA translational repression. Mutation in the N-terminal zinc-binding domain of A3G important for virion incorporation and mutation in the C-terminal zinc-binding domain important for cytidine deaminase activity were examined for their possible influence on miRNA-mediated translational repression (28–31). The mutations that inactivate the N-terminal domain, C97A and C100A, had a modest effect on miRNA-mediated translational repression, whereas the C-terminal domain C288A and C291A mutations had no significant influence on the inhibitory effect of A3G (Fig. 3b), suggesting that the cytidine deaminase activity is unlikely involved in this effect.

A3G Enhances the Association of miRNA-targeted mRNA with Polysomes—To examine whether the A3G inhibitory effect on mir-16-mediated repression was at the level of translation, a polysome profile analysis was performed (Fig. 4). As shown in Fig. 4c, mir-16 decreased the association of its target mRNA with polysomes, which is consistent with previous reports (45, 46). However, A3G, as well as an antisense anti-mir-16 inhibitor, significantly enhanced the association of the target mRNA with polysomes (Fig. 4, d and e). Puromycin treatment can disrupt this association, further confirming the complex that luciferase mRNA bound with is polysome (Fig. 4g).

A3G Facilitates the Dissociation of miRNA-targeted mRNA from P-bodies—As A3G can be found in P-bodies (32, 33), and can increase the amount of miRNA-targeted mRNA (Fig. 1e), we then investigated whether A3G could be directly associated with GW182, a key component for P-body. We found that A3G can interact with GW182. This interaction is partially resistant to RNase digestion. Mutation at C-terminal catalytic domain of A3G (C288A/C291A) cannot eliminate this interaction (Fig. 5a). Further, we also confirmed that A3G co-localized with GW182 (Fig. 5b) (32, 33). Moreover, we have found that the depletion of GW182 with GW182-specific siRNA had a synergistic effect with A3G in counteracting miRNA-mediated translational repression (Fig. 5c), which is consistent with previous reports regarding the role of GW182 in miRNA function (15, 16).

We then examined whether A3G had any effect on the interaction between miRNA-targeted mRNA and P-bodies by performing in situ hybridization with confocal microscopy, as described (21). The location of luciferase mRNA was detected with a Cy3-conjugated oligonucleotide probe, and the location of P-bodies was visualized with GFP-GW182 (19). The mRNA without miRNA-binding sites did not associate with GW182 (Fig. 6, a and b). In the absence of A3G, mir-16-targeted luciferase mRNA was found associated with GW182 and in P-bodies (Fig. 6c), indicating that miRNAs such as mir-16 mediate the association of mRNA with P-bodies. However, in the presence of A3G, mir-16-targeted luciferase mRNA was not found in the P-body (Fig. 6d), suggesting that A3G either facilitates the exit of miRNA-bound mRNA from P-bodies or prevents miRNA-bound mRNA from entering P-bodies. As a control, an anti-mir-16 antisense inhibitor, which can specifically block the function of mir-16, but not an anti-mir28 inhibitor, also prevented the miRNA-targeted luciferase mRNA from associating with GW182 and P-bodies (Fig. 6, e and f).

Interaction between A3G and GW182.a, 293T cells were transfected with pcDNA3-A3G-HA or pcDNA3-A3G-C97A/C100A-HA. At 48-h post-transfection, cells were collected and lysed. Lysates were treated with and without RNase A, followed by immunoprecipitation with mouse anti-GW182 antibody. The precipitated samples were then subjected to SDS-PAGE electrophoresis. After transferring, A3G was detected with rabbit anti-A3G antibody. b, HeLa cells were co-transfected with pcDNA-A3G-HA and pGFP-GW182delta1 (19). At 48-h post-transfection, the localization of GW182 was visualized with GFP-GW182 fluorescence (green) and A3G was detected with mouse anti-A3G and visualized with Texas Red-conjugated goat anti-mouse antibody (red). c, 293T cells were transfected with GW182-specific siRNA. At 48-h post-transfection, the cells were co-transfected with pcDNA-A3G-HA and pmir16-luc. After another 48 h, a luciferase assay was performed. The means ± S.D. are shown.

A3G facilitates the dissociation of mir-16-targeted mRNA from P-bodies. HeLa cells were co-transfected with pcDNA-A3G-HA, pmir16-luc, pGFP-GW182delta1, or various antisense miRNA inhibitors, as indicated. At 48-h post-transfection, P-bodies were visualized with GFP-GW182 fluorescence (green), and luciferase mRNA was visualized by in situhybridization with Cy3-conjugated oligonucleotide probes (red). DAPI staining of the nuclei is shown inblue. A magnification of the regions enclosed by the boxes is shown in the insets at the upper left corners.

Discussion

Endogenous A3G can be found in various cells such as H9 T-cells, primary CD4 T-cells, macrophages, and many other normal tissues/organs such as spleen, thymus, testis, ovary, small intestine, mucosal lining of colon (22, 47). They can effectively inhibit the replication of vif-defective HIV-1 (22, 48, 49). Although miRNAs are still able to mediate translational inhibition in H9 T-cells, primary CD4 T-cells at a moderate level and in macrophage at a significant level, we believe that their activity has been restricted by endogenous A3G/A3F. As shown in Fig. 2, a-c, A3G/F-specific siRNAs, which effectively deplete A3G/F in these cells, can significantly further enhance the miRNA-mediated translational inhibition, indicating endogenous A3G or A3F are functional to prevent the activity of miRNA. Furthermore, overexpression of A3G/F can effectively counteract the miRNA-mediated inhibitory effect on translation, supporting this argument (Fig. 2c). Nevertheless, the result from overexpression of exogenous A3G/F also suggests that the either quantity or quality of endogenous A3G/F could need to be improved for an efficient counteraction to miRNA activity. Recently, we and others have found that interferon-(IFN)-α/β can significantly enhance the expression of A3G/F in various primary cells such as resting CD4 T-lymphocytes, macrophages, endothelial cells, hepatocytes, myeloid dendritic cells, and plasmacytoid dendritic cells (42, 50–54).3 Therefore, it is interesting to further investigate the correlation of IFN regulatory system and the miRNA activity in these primary cells.

Our data demonstrate that A3G facilitates recruitment of miRNA-targeted mRNA to polysomes to synthesize more proteins and drives dissociation of miRNA-targeted mRNA from P-bodies. Given that A3G is associated with mRNA, localizes to P-bodies and stress granules (32, 33, 36), and can substantially enhance the expression of miRNA-targeted mRNA (Fig. 1e), it is unlikely that A3G directly improves the interaction between mRNA and polysomes or inhibits the interaction between miRNA and its target mRNA in miRISC. Instead, A3G may block miRNA-targeted mRNA from entering P-bodies or stress granules, may prevent the miRNA-targeted mRNA from engaging the RNA degradation machinery in P-bodies, or may directly facilitate the egress of miRNA-targeted mRNA from P-bodies and stress granules. By one or more of these approaches, A3G may inhibit the degradation or storage of miRNA-targeted miRNA in P-bodies and stress granules. Subsequently, more of the mRNA could associate with polysomes, and the translation efficiency would therefore be enhanced. However, as the mechanism of the regulation of mRNA degradation and storage in P-bodies or stress granules remains to be clarified and the relationship between miRNA-mediated translational repression and P-bodies is still under intensive investigation, further experiments are required to demonstrate the exact mechanism underlying this cellular function of A3G.

Interestingly, the mutations C228A and C291A inactivated the cytidine deaminase activity of A3G, but A3G was still able to enhance the expression of luciferase when luc was controlled by miRNA (Fig. 3b). Therefore, the derepression of miRNA-mediated inhibition of protein translation by A3G is separable from its cytidine deaminase activity. As described in many reports, the cytidine deaminase activity of A3G is only partially responsible for viral infectivity (24, 28–31). It remains to be determined whether this cellular function of A3G in protein translation regulation is related to its cytidine deaminase-independent antiviral activity.

↵* This work was supported in part by National Institutes of Health Grants AI058798 and AI052732 (to H. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵ The on-line version of this article (available athttp://www.jbc.org) contains supplemental Figs. S1-S3 and Table S1.

In a tumor cell, a mutation in the Bcl-2 gene results in increased expression will suppress the normal function of the pro-apoptotic proteins BAX and BAK, leading to malignancy. On the other hand, a mutation in the BAX or BAK genes can cause a down-regulation of expression, causing the cell to lose the ability to regulate apoptosis, once again, leading to cancer cells. The inhibitor of apoptosis (IAP) family genes, which encode negative regulatory proteins, can prevent apoptotic cell death.
In the normal cell, the p53 protein binds DNA, stimulating another gene to produce a protein called p21, which interacts with a cell division stimulating protein (cdk2) [11]. When p21 forms a complex with cdk2, the cell cannot pass through to the next stage of cell division, and remains arrested in G1 [7]. The p53 protein product of a TP53 mutant gene cannot bind DNA in an effective way, and as a consequence, the p21 protein is not made available to act as the stop signal for the cell cycle/cell division. Therefore, cells divide uncontrollably and form tumors [4] Not surprisingly, there is an increased frequency in the amplification of the ubiquitin ligases protein (MDM2) involved in the mechanism for the down regulation of p53 activity through ubiquitin-dependent proteosomal degradation of p53 [36].
P53 has been shown to promote hematopietic stem cells (HSCs) quiescence and self-renewal with recent studies showing that deficiency of p53 likely promotes acute myeloid leukemia (AML) by eliminating its ability to limit aberrant self-renewal in hematopoietic progenitors. Micro RNAs (miRNAs) are small non-protein-coding RNAs that regulate gene expression by inhibiting the translation or catalyzing the degradation of target mRNAs. Since the first miRNA, lin-4, was identified in 1993, miRNAs have been shown to play critical roles in the regulation of many biological processes including cell differentiation, proliferation, and apoptosis, with significant influences on normal and malignant hematopoiesis [32].

The majority of living forms depend for their functioning upon two classes of biocatalysts, the enzymes and the hormones. These biocatalysts permit the diverse chemical reactions of the organism to proceed at 38°C with specificity and at rates frequently unattainable in vitro at elevated temperatures with similar reactants. The physiologic importance of enzymes and hormones is evident not only under normal circumstances, but is reflected clinically in the diverse descriptions of errors of metabolism, due to lack or deficiency of one or more enzymes, and the numerous hypo and hyper functioning states resulting from imbalance of hormonal supply.

In as much as both enzymes and hormones function, with rare exception, to accelerate the rates of processes in cells, investigators have sought possible interrelationships and interactions of enzymes and hormones, particularly as a basis for the mechanism of hormonal action. It has seemed logical to hypothesize that hormones, while not essential for reactions to proceed but never the less affecting the rates of reactions, may function by altering either the concentration or activity of the prime cellular catalysts, the enzymes. This proposed influence of hormones on enzymatic activity might be a primary, direct effect achieved by the hormone participating as an integral part of an enzyme system, or an indirect influence based upon the hormone altering the concentration of available enzyme and/or substrate utilized by a particular enzyme. Many publications have described alterations in the activity of enzymes in various tissues following administration in vivo of diverse hormonal preparations. However, it is not possible to judge, in the in vivo experiments, whether the reported effects are examples of direct enzyme-hormone interaction, or an indirect influence of the hormone mediated via one or more metabolic pathways, and therefore other enzyme systems whose activities are not being measured. Data from in-vivo studies of this type are thus not pertinent to a discussion of direct hormone-enzyme interaction.

Enzyme hormone interaction, as seen, for example, in the profound role of the enzymes of the liver in the metabolism of certain hormones, is of paramount importance in determining the effectiveness of these hormones. The ability of the organic chemist to prepare synthetic hormonal derivatives which are relatively resistant to enzymatic processes in the liver has been of outstanding value for approaches to oral hormonal therapy. Largely unexplored as yet is the possibility that enzyme-hormone interactions may lead to the production of physiologically more active substances from compounds normally synthesized and secreted by a particular endocrine gland. It may be said at the outset that in no instance has a hormone been demonstrated to influence the rate of a cellular reaction by functioning as a component of an enzyme system.

It is plausible that enzymes in a pathway might be structurally conserved because of their similar substrates and products for linked metabolic steps. However, this is not typically observed, and sequence analysis confirms the lack of convergent or divergent evolution. One might postulate that, if the folds or overall structures of the enzymes in a pathway are not conserved, then perhaps at least pathway-related active site similarities would exist. It is true that metal-binding sites and nucleotide-binding sites are structurally conserved. For example, cofactor-binding motifs for zinc, ATP, biopterin and NAD have been observed and biochemically similar reactions appear to maintain more structural similarity than pathway-related structural motifs. In general, ‘horizontal’ structural equivalency is prevalent in that chemistry-related structural similarities exist, but ‘vertical’ pathway-related structural similarities do not hold.

For metabolic pathways, protein fold comparisons and corresponding active site comparisons are sometimes possible if structural and functional homology exists. Unfortunately, with the current structural information available, the majority of active sites that can be structurally characterized are not similar within a metabolic pathway. Other examples exist of nearly completed pathways, for example, the tricarboxylic acid (TCA) cycle, and similar observations are observed. Situations in which different metals are incorporated in enzyme active sites lead to inherently different catalytic portions of the active sites. Slight differences in the ligand-binding portions of the respective active sites must lead to the observed differences in pathway-related enzyme specificities. These modifications in enzymatic activity are similar to what Koshland and co-workers previously observed. They showed that very minor active site perturbations to isocitrate dehydrogenase had drastic effects on catalysis.

Molecular level understanding of chemical and biological processes requires mechanistic details and active site information. The current knowledge regarding enzyme active sites is incomplete. Even in situations in which ATP-, ADP- or NAD(P)+-binding domains are observed or in situations in which similar folds are found (e.g. even for related kinases or for proteins involved in the immune system), structural comparisons do not yield specific details about active sites and it is not possible to predict where the substrate binds or to identify determinants of active site substrate specificity. Therefore, in this era of structural genomics, there should be major continued emphasis on completing structural information for important metabolic pathways. This will require improved efforts to obtain structures for enzyme complexes with appropriate cofactors, substrates or substrate analogs, as well as with inhibitors and regulators of activity. Then and only then will we have complete structural knowledge and facilitated structure-based drug design efforts. Structural genomics efforts promise to provide structural data in a high-throughput mode. However, we need to ensure that much of this focus is placed on completing the picture of metabolic pathways and enzyme active sites.

The availability of the human genomic sequence is changing the way in which biological questions are addressed. Based on the prediction of genes from nucleotide sequences, homologies among their encoded amino acids can be analyzed and used to place them in distinct families. This serves as a first step in building hypotheses for testing the structural and functional properties of previously uncharacterized paralogous genes. As genomic information from more organisms becomes available, these hypotheses can be refined through comparative genomics and phylogenetic studies. Instead of the traditional single-gene approach in endocrine research, we are beginning to gain an understanding of entire mammalian genomes, thus providing the basis to reveal subfamilies and pathways for genes involved in ligand signaling. The present review provides selective examples of postgenomic approaches in the analysis of novel genes involved in hormonal signaling and their chromosomal locations, polymorphisms, splicing variants, differential expression, and physiological function. In the postgenomic era, scientists will be able to move from a gene-by-gene approach to a reconstructionistic one by reading the encyclopedia of life from a global perspective. Eventually, a community-based approach will yield new insights into the complexity of intercellular communications, thereby offering us an understanding of hormonal physiology and pathophysiology. Many cellular signaling pathways ultimately control specific patterns of gene expression in the nucleus through a variety of signal-regulated transcription factors, including nuclear hormone receptors. The advent of genomic technologies for examining signal-regulated transcriptional responses and transcription factor binding on a genomic scale has dramatically increased our understanding of the cellular programs that control hormonal signaling and gene regulation. Studies of transcription factors, especially nuclear hormone receptors, using genomic approaches have revealed novel and unexpected features of hormone-regulated transcription, and a global view is beginning to emerge.